Aromaticity at spike position 456 affects functional immunogenicity of SARS-CoV-2 Omicron variant receptor-binding domain

The recurrent emergence of the F456L substitution in SARS-CoV-2 Omicron sublineages has been associated with immune evasion, yet its impact on vaccine-induced immune responses remains poorly understood. Using a trimeric RBD-HR subunit vaccine platform, we investigated how residue 456 influences the functional immunogenicity of the receptor-binding domain (RBD). We found that the F456L substitution markedly reduced neutralizing antibody responses against multiple SARS-CoV-2 variants, despite maintaining comparable levels of total antigen-specific IgG, indicating a selective impairment of functional immunogenicity rather than overall antigenicity. Single-cell RNA sequencing and B-cell receptor (BCR) repertoire analyses further revealed that the F456L mutation weakened germinal center responses, reduced clonal expansion, and impaired repertoire remodeling of antigen-specific B cells. Structural modeling, biolayer interferometry, and calcium flux assays demonstrated that aromatic residues at position 456 promote effective germline BCR recognition and activation. Importantly, replacing the non-aromatic leucine residue with aromatic amino acids, including tyrosine or tryptophan, restored the neutralizing antibody-eliciting capacity of the antigen. Together, these findings identify aromaticity at residue 456 as a critical determinant of RBD functional immunogenicity and demonstrate how a single amino acid substitution can reshape B-cell responses and antibody quality. This work provides important insights for rational vaccine antigen design and advances our understanding of how site-specific mutations regulate antiviral immunity.